Postoperative Changes in Amniotic Membrane as a Carrier for Allogeneic Cultured Limbal Epithelial Transplantation XIAOLIN QI, JUNYI WANG, DAPENG SUN, QINGJUN ZHOU, AND LIXIN XIE PURPOSE:
To investigate the morphologic changes and outcomes of the amniotic membrane as a carrier for allogeneic cultivated limbal epithelial transplantation. DESIGN: Prospective, noncomparative, interventional study. METHODS: A total of 16 eyes receiving allogeneic cultivated limbal epithelial transplantation with amniotic membrane as a carrier were enrolled. Morphologic changes in the amniotic membrane were observed by confocal microscopy and RTVue optical coherence tomography. The paired t test was employed to compare the mean best corrected visual acuity (BCVA) and corneal stromal thickness. RESULTS: Of the 16 eyes, 12 had stable ocular surfaces (group A), while the other 4 eyes had failed surgeries due to immune rejection (group B). Confocal microscopy showed residual amniotic membrane tissues in 8 eyes in group A at 1 year. However, the amniotic membrane was not detected in group B at 8–10 months. RTVue optical coherence tomography showed discontinuous amniotic membrane tissues in all eyes in group A at 1 year, while highly reflective opacity was seen in the corneal stroma in group B. There were no statistically significant differences in mean BCVA and corneal stromal thickness in group A at 1 month and 1 year after transplantation (P > 0.05), but the mean BCVA showed a statistically significant difference at 1 month and after the disappearance of the amniotic membrane in group B (P < 0.05). CONCLUSIONS: For eyes with stable ocular surfaces after cultivated limbal epithelial transplantation, the amniotic membrane can be present in the cornea for at least 1 year, with no impact on visual acuity or corneal stromal thickness. Chronic inflammation and neovascularization on the ocular surface may accelerate the disappearance of the amniotic membrane. (Am J Ophthalmol 2014;158:1192–1198. Ó 2014 by Elsevier Inc. All rights reserved.)
A
LLOGENEIC
CULTIVATED
LIMBAL
EPITHELIAL
transplantation has been found to be effective in the treatment of ocular chemical and thermal
Accepted for publication Aug 12, 2014. From the Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China. Inquiries to Lixin Xie, Shandong Eye Institute, 5 Yanerdao Road, Qingdao 266071, China; e-mail: [email protected]
1192
Ó
2014 BY
burns.1–3 The amniotic membrane seems to be an ideal vehicle for the in vitro cultivation and transplantation of limbal epithelial stem cells4,5 because of its unique biologic properties.6,7 However, neither the morphologic changes and final outcomes of the amniotic membrane on the ocular surface nor their impact on visual acuity, corneal stromal thickness and transparency after transplantation have been reported. In this prospective study, we enrolled patients who had received cultivated limbal epithelial transplantation for ocular chemical or thermal injuries and observed the changes in the amniotic membrane on the ocular surface postoperatively by various means, including confocal microscopy and corneal optical coherence tomography (OCT).
METHODS PATIENTS: This prospective, noncomparative, interventional study was approved by the Institutional Review Board of Shandong Eye Institute and adhered to the tenets of the Declaration of Helsinki. Patients undergoing allogeneic cultivated limbal epithelial transplantation with human amniotic membrane as a carrier at Shandong Eye Institute were recruited on a consecutive basis from January-April 2012. The eligible eyes met the following inclusion criteria: (1) total limbal stem cell deficiency arising from ocular chemical or thermal burns was diagnosed if (a) clinical slit-lamp examination revealed 360-degree loss of limbal palisades of Vogt, chronic ocular surface inflammation, poor epithelial integrity, corneal conjunctivalization, and neovascularization; and (b) corneal impression cytology showed the presence of goblet cells8,9; (2) allogeneic cultivated limbal epithelial transplantation was performed at least 6 months after ocular burns; (3) no keratoplasty, limbal tissue transplantation or cultivated limbal epithelial transplantation had been performed previously; (4) a follow-up of at least 12 months was completed at Shandong Eye Institute. Patients with severe dry eye (Schirmer test 0.05). MEAN BCVA:
There was a statistically significant difference in the mean BCVA between the before-surgery levels (0.02; range, 0.01–0.05) and at 1 month (0.12; range, 0.06–0.15) (P < 0.05) in group A. The mean BCVA was 0.15 (range, 0.06–0.20) at 1 year, with no significant difference from that at 1 month (P > 0.05). The mean BCVA in group B was significantly better than the preoperative 0.02 (range, 0.01–0.05) to 0.1 (range, 0.05–0.12) at 1 month after transplantation (P < 0.05). However, the mean BCVA decreased to 0.02 (range, 0.01–0.08) after the amniotic membrane disappeared, significantly different from that at 1 month (P < 0.05).
DISCUSSION AMNIOTIC MEMBRANE TRANSPLANTATION CAN PLAY A
role in the treatment of infective keratitis and other ocular diseases.5,6 In general, 3 surgical approaches are available:
AMNIOTIC MEMBRANE IN LIMBAL EPITHELIAL TRANSPLANTATION
1195
FIGURE 3. Corneal optical coherence tomography after cultured limbal epithelial transplantation using the amniotic membrane as a carrier. (Top left) At 1 month after transplantation, the amniotic membrane beneath the corneal epithelium presented a continuous, reflectively enhanced arclike structure with an even thickness of approximately 23.2 ± 3.4 mm and distinct borders with the epithelial layer and the stromal layer. (Top right) The amniotic membrane gradually grew thinner in group A and became discontinuous from the peripheral cornea at 4 months. (Bottom left) At 1 year, discontinuous, reflectively enhanced amniotic membrane tissues were observed in all 8 eyes of group A. (Bottom right) Immune rejection caused opacity in the corneal stroma, with highly reflective structures, and the amniotic membrane tissues were not recognizable.
patch, graft and sandwich,17,18 with different morphologic changes and outcomes after the amniotic membrane is transplanted onto the ocular surface. Amniotic membrane is used after 2 weeks of cultivation as a carrier in cultivated limbal epithelial transplantation, with 3–5 layers of cell films on its surface.13 Whether there are dynamic changes in the amniotic membrane after its transplantation and whether unique objective principles specific to the above surgical approaches are involved in these changes have been unknown and are focused on in this prospective study. The ocular surface reconstruction was successful in 12 eyes but failed in 4 eyes due to immune rejection. Differing characteristics were found in the 2 groups of subjects. Among the 12 eyes achieving ocular surface reconstruction, unabsorbed amniotic membrane tissues were observed by confocal microscopy and corneal OCT at 1 year after cultivated limbal epithelial transplantation. The amniotic membrane can function as the basal membrane in cultivated limbal epithelial transplantation, and the corneal epithelial layer migrates on its surface. As in the ‘‘graft’’ transplantation method, the amniotic membrane is covered by the epithelial layer after epithelialization.18 As suggested by Tsai et al,19 the amniotic membrane has a slow absorption rate when this transplantation method is used. By electronic microscopy and histologic examination, Resch et al20 discovered that the amniotic membrane persisted in the corneal stroma for up to 79 months. Whether the amniotic membrane can exist in the corneal stroma for a longer period and the time of its final disappearance after cultivated limbal epithelial 1196
transplantation need further follow-up. In contrast, the amniotic membrane could not be observed at 8–10 months after cultivated limbal epithelial transplantation in the 4 eyes suffering immune rejection in our series. Confocal microscopy showed a large amount of inflammatory cell infiltration in the corneal stroma beneath the amniotic membrane, but this abnormality was not found in eyes with stable ocular surfaces. Therefore, we hypothesized that the chronic inflammation on the ocular surface caused by immune rejection was an important factor in accelerating the disappearance of the amniotic membrane.21–25 The amniotic membrane is translucent. Gris et al17 believed that the persistent presence of the amniotic membrane in the corneal stroma affected corneal transparency. In this study, at 7–10 days after cultivated limbal epithelial transplantation, the amniotic membrane was grayish-white and cloudy and adhered tightly to the corneal surface. Due to the combination of surgical stimulation and inflammation, the corneal stroma exhibited edema, cloudiness and decreased transparency. At 1 month, no amniotic membrane tissues but subepithelial ground-glass opacity was observed on the ocular surface. The stromal edema had subsided, showing improved transparency. The BCVA was significantly improved. At 2 months, fine gridlike cloudiness was detected beneath the corneal epithelium in patients with stable ocular surfaces, and it gradually faded during the follow-up period. The homogeneous, reflectively enhanced acellular structure, which was the unabsorbed amniotic membrane tissues, was visible only by confocal microscopy at 1 year. The BCVA was not
AMERICAN JOURNAL OF OPHTHALMOLOGY
DECEMBER 2014
significantly different from that at 1 month. This indicates that the presence of stable amniotic membrane exerts no effect on the corneal stromal transparency or visual acuity. In contrast, corneal cloudiness and neovascularization occurred again in the 4 eyes suffering immune rejection. The BCVA substantially decreased during the follow-up. This suggests that much attention is needed to prevent and treat immune rejection after cultivated limbal epithelial transplantation.14 In infective corneal diseases, the transplantation of a multilayer amniotic membrane can increase the thickness of the corneal stroma and avoid complications such as ulcer perforation.26–30 In this study, corneal OCT showed gradual thinning of the amniotic membrane in the 12
eyes with stable ocular surfaces. Discontinuity of the amniotic membrane was observed at 4 months after cultivated limbal epithelial transplantation and became more evident at 1 year. The corneal stromal thickness did not change significantly. This is most likely related to the use of the single-layer, thin amniotic membrane. Amniotic membrane is an ideal vehicle for the in vitro cultivation and transplantation of limbal epithelial stem cells. It can survive for at least 1 year in the cornea of a patient with a stable ocular surface, without impact on the visual acuity, corneal stromal transparency or thickness. Chronic inflammation and neovascularization of the ocular surface can accelerate the absorption of the amniotic membrane and lead to decreased corneal transparency.
ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST, and the following were reported. This study was supported by the National Basic Research Program of China (2013CB967004) and the Specialized Research Fund for the Doctoral Program of Higher Education (20113706110004). Involved in Design and conduct of study (X.Q.); Collection of data (J.W., D.S.); Management, analysis and interpretation of data (X.Q., J.W., Q.Z.); Preparation of manuscript (X.Q.); Review and approval of manuscript (L.X.). The authors thank Ping Lin, Shandong Eye Institute, for her linguistic assistance.
REFERENCES 1. Koizumi N, Inatomi T, Suzuki T, Sotozono C, Kinoshita S. Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology 2001;108(9): 1569–1574. 2. Koizumi N, Inatomi T, Suzuki T, Sotozono C, Kinoshita S. Cultivated corneal epithelial transplantation for ocular surface reconstruction in acute phase of Stevens-Johnson syndrome. Arch Ophthalmol 2001;119(2):298–300. 3. Nakamura T, Sotozono C, Bentley AJ, et al. Long-term phenotypic study after allogeneic cultivated corneal limbal epithelial transplantation for severe ocular surface diseases. Ophthalmology 2010;117(12):2247–2254. 4. Dua HS, Gomes JA, King AJ, Maharajan VS. The amniotic membrane in ophthalmology. Surv Ophthalmol 2004;49(1): 51–77. 5. Koizumi NJ, Inatomi TJ, Sotozono CJ, Fullwood NJ, Quantock AJ, Kinoshita S. Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res 2000; 20(3):173–177. 6. Meller D, Pires RT, Tseng SC. Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures. Br J Ophthalmol 2002;86(4): 463–471. 7. Burman S, Tejwani S, Vemuganti GK, Gopinathan U, Sangwan VS. Ophthalmic applications of preserved human amniotic membrane: a review of current indications. Cell Tissue Bank 2004;5(3):161–175. 8. Dua HS, Joseph A, Shanmuganathan VA, Jones RE. Stem cell differentiation and the effects of deficiency. Eye 2003; 17(8):877–885. 9. Shortt AJ, Secker GA, Notara MD, et al. Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results. Surv Ophthalmol 2007;52(5): 483–502.
VOL. 158, NO. 6
10. Lee S, Tseng SC. Amniotic membrane transplantation for persistent epithelial defects with ulceration. Am J Ophthalmol 1997;123(3):303–312. 11. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000;343(2):86–93. 12. Nishida K, Yamato M, Hayashida Y, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 2004; 351(12):1187–1196. 13. Zhou Q, Yang L, Wang Y, et al. TGFbeta mediated transition of corneal fibroblasts from a proinflammatory state to a profibrotic state through modulation of histone acetylation. J Cell Physiol 2010;224(1):135–143. 14. Qi X, Xie L, Cheng J, Zhai H, Zhou Q. Characteristics of immune rejection after allogeneic cultivated limbal epithelial transplantation. Ophthalmology 2013;120(5):931–936. 15. Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 2010;363(2):147–155. 16. Chen Y, Yan XM, Wu HR, Rong B. An experimental study on the fate of the amniotic membrane after amniotic membrane transplantation for acute alkaline burn of rat cornea. Zhonghua Yan Ke Za Zhi 2012;48(1):27–32. 17. Gris O, Wolley-Dod C, Gu¨ell JL, et al. Histologic findings after amniotic membrane graft in the human cornea. Ophthalmology 2002;109(3):508–512. 18. Maharajan VS, Shanmuganathan V, Currie A, Hopkinson A, Powell-Richards A, Dua HS. Amniotic membrane transplantation for ocular surface reconstruction: indications and outcomes. Clin Exp Ophthalmol 2007;35(2):140–147. 19. Tsai RJ, Tsai RY. Ex vivo expansion of corneal stem cells on amniotic membrane and their outcome. Eye Contact Lens 2010;36(5):305–309. 20. Resch MD, Schlo¨tzer-Schrehardt U, Hofmann-Rummelt C, et al. Integration patterns of cryopreserved amniotic
AMNIOTIC MEMBRANE IN LIMBAL EPITHELIAL TRANSPLANTATION
1197
21.
22. 23.
24.
25.
membranes into the human cornea. Ophthalmology 2006; 113(11):1927–1935. Gris O, Lo´pez-Navidad A, Caballero F, del Campo Z, Ada´n A. Amniotic membrane transplantation for ocular surface pathology: long-term results. Transplant Proc 2003;35(5): 2031–2035. Bourne WM. Cellular changes in transplanted human corneas. Cornea 2001;20(6):560–569. Hollingsworth JG, Efron N, Tullo AB. A longitudinal case series investigating cellular changes to the transplanted cornea using confocal microscopy. Cont Lens Anterior Eye 2006; 29(3):135–141. Chen M, Gong L, Xu J, Zhu W, Devine EE. Ultrastructural and in vivo confocal microscopic evaluation of interface after Descemet’s stripping endothelial keratoplasty in rabbits. Acta Ophthalmol 2012;90(1):e43–47. Abdelkader A, Kaufman HE. Descemetic versus preDescemetic lamellar keratoplasty: clinical and confocal study. Cornea 2011;30(11):1244–1252.
1198
26. Tosi GM, Traversi C, Schuerfeld K, et al. Amniotic membrane graft: histopathological findings in five cases. J Cell Physiol 2005;202(3):852–857. 27. Letko E, Stechschulte SU, Kenyon KR, et al. Amniotic membrane inlay and overlay grafting for corneal epithelial defects and stromal ulcers. Arch Ophthalmol 2001;119(5): 659–663. 28. Said DG, Nubile M, Alomar T, et al. Histologic features of transplanted amniotic membrane: implications for corneal wound healing. Ophthalmology 2009;116(7): 1287–1295. 29. Resch MD, Schlo¨tzer-Schrehardt U, Hofmann-Rummelt C, et al. Adhesion structures of amniotic membranes integrated into human corneas. Invest Ophthalmol Vis Sci 2006;47(5): 1853–1861. 30. Stoiber J, Muss WH, Pohla-Gubo G, Ruckhofer J, Grabner G. Histopathology of human corneas after amniotic membrane and limbal stem cell transplantation for severe chemical burn. Cornea 2002;21(5):482–489.
AMERICAN JOURNAL OF OPHTHALMOLOGY
DECEMBER 2014
Biosketch Dr Xiaolin Qi is an attending ophthalmologist at the Shandong Eye Hospital of Shandong Eye Institute. She received her master’s degree and doctoral degree in ophthalmology from Qingdao University. Dr Qi is engaged in clinical work of corneal disease.
VOL. 158, NO. 6
AMNIOTIC MEMBRANE IN LIMBAL EPITHELIAL TRANSPLANTATION
1198.e1
To investigate the morphologic changes and outcomes of the amniotic membrane as a carrier for allogeneic cultivated limbal epithelial transplantation. DESIGN: Prospective, noncomparative, interventional study. METHODS: A total of 16 eyes receiving allogeneic cultivated limbal epithelial transplantation with amniotic membrane as a carrier were enrolled. Morphologic changes in the amniotic membrane were observed by confocal microscopy and RTVue optical coherence tomography. The paired t test was employed to compare the mean best corrected visual acuity (BCVA) and corneal stromal thickness. RESULTS: Of the 16 eyes, 12 had stable ocular surfaces (group A), while the other 4 eyes had failed surgeries due to immune rejection (group B). Confocal microscopy showed residual amniotic membrane tissues in 8 eyes in group A at 1 year. However, the amniotic membrane was not detected in group B at 8–10 months. RTVue optical coherence tomography showed discontinuous amniotic membrane tissues in all eyes in group A at 1 year, while highly reflective opacity was seen in the corneal stroma in group B. There were no statistically significant differences in mean BCVA and corneal stromal thickness in group A at 1 month and 1 year after transplantation (P > 0.05), but the mean BCVA showed a statistically significant difference at 1 month and after the disappearance of the amniotic membrane in group B (P < 0.05). CONCLUSIONS: For eyes with stable ocular surfaces after cultivated limbal epithelial transplantation, the amniotic membrane can be present in the cornea for at least 1 year, with no impact on visual acuity or corneal stromal thickness. Chronic inflammation and neovascularization on the ocular surface may accelerate the disappearance of the amniotic membrane. (Am J Ophthalmol 2014;158:1192–1198. Ó 2014 by Elsevier Inc. All rights reserved.)
A
LLOGENEIC
CULTIVATED
LIMBAL
EPITHELIAL
transplantation has been found to be effective in the treatment of ocular chemical and thermal
Accepted for publication Aug 12, 2014. From the Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China. Inquiries to Lixin Xie, Shandong Eye Institute, 5 Yanerdao Road, Qingdao 266071, China; e-mail: [email protected]
1192
Ó
2014 BY
burns.1–3 The amniotic membrane seems to be an ideal vehicle for the in vitro cultivation and transplantation of limbal epithelial stem cells4,5 because of its unique biologic properties.6,7 However, neither the morphologic changes and final outcomes of the amniotic membrane on the ocular surface nor their impact on visual acuity, corneal stromal thickness and transparency after transplantation have been reported. In this prospective study, we enrolled patients who had received cultivated limbal epithelial transplantation for ocular chemical or thermal injuries and observed the changes in the amniotic membrane on the ocular surface postoperatively by various means, including confocal microscopy and corneal optical coherence tomography (OCT).
METHODS PATIENTS: This prospective, noncomparative, interventional study was approved by the Institutional Review Board of Shandong Eye Institute and adhered to the tenets of the Declaration of Helsinki. Patients undergoing allogeneic cultivated limbal epithelial transplantation with human amniotic membrane as a carrier at Shandong Eye Institute were recruited on a consecutive basis from January-April 2012. The eligible eyes met the following inclusion criteria: (1) total limbal stem cell deficiency arising from ocular chemical or thermal burns was diagnosed if (a) clinical slit-lamp examination revealed 360-degree loss of limbal palisades of Vogt, chronic ocular surface inflammation, poor epithelial integrity, corneal conjunctivalization, and neovascularization; and (b) corneal impression cytology showed the presence of goblet cells8,9; (2) allogeneic cultivated limbal epithelial transplantation was performed at least 6 months after ocular burns; (3) no keratoplasty, limbal tissue transplantation or cultivated limbal epithelial transplantation had been performed previously; (4) a follow-up of at least 12 months was completed at Shandong Eye Institute. Patients with severe dry eye (Schirmer test 0.05). MEAN BCVA:
There was a statistically significant difference in the mean BCVA between the before-surgery levels (0.02; range, 0.01–0.05) and at 1 month (0.12; range, 0.06–0.15) (P < 0.05) in group A. The mean BCVA was 0.15 (range, 0.06–0.20) at 1 year, with no significant difference from that at 1 month (P > 0.05). The mean BCVA in group B was significantly better than the preoperative 0.02 (range, 0.01–0.05) to 0.1 (range, 0.05–0.12) at 1 month after transplantation (P < 0.05). However, the mean BCVA decreased to 0.02 (range, 0.01–0.08) after the amniotic membrane disappeared, significantly different from that at 1 month (P < 0.05).
DISCUSSION AMNIOTIC MEMBRANE TRANSPLANTATION CAN PLAY A
role in the treatment of infective keratitis and other ocular diseases.5,6 In general, 3 surgical approaches are available:
AMNIOTIC MEMBRANE IN LIMBAL EPITHELIAL TRANSPLANTATION
1195
FIGURE 3. Corneal optical coherence tomography after cultured limbal epithelial transplantation using the amniotic membrane as a carrier. (Top left) At 1 month after transplantation, the amniotic membrane beneath the corneal epithelium presented a continuous, reflectively enhanced arclike structure with an even thickness of approximately 23.2 ± 3.4 mm and distinct borders with the epithelial layer and the stromal layer. (Top right) The amniotic membrane gradually grew thinner in group A and became discontinuous from the peripheral cornea at 4 months. (Bottom left) At 1 year, discontinuous, reflectively enhanced amniotic membrane tissues were observed in all 8 eyes of group A. (Bottom right) Immune rejection caused opacity in the corneal stroma, with highly reflective structures, and the amniotic membrane tissues were not recognizable.
patch, graft and sandwich,17,18 with different morphologic changes and outcomes after the amniotic membrane is transplanted onto the ocular surface. Amniotic membrane is used after 2 weeks of cultivation as a carrier in cultivated limbal epithelial transplantation, with 3–5 layers of cell films on its surface.13 Whether there are dynamic changes in the amniotic membrane after its transplantation and whether unique objective principles specific to the above surgical approaches are involved in these changes have been unknown and are focused on in this prospective study. The ocular surface reconstruction was successful in 12 eyes but failed in 4 eyes due to immune rejection. Differing characteristics were found in the 2 groups of subjects. Among the 12 eyes achieving ocular surface reconstruction, unabsorbed amniotic membrane tissues were observed by confocal microscopy and corneal OCT at 1 year after cultivated limbal epithelial transplantation. The amniotic membrane can function as the basal membrane in cultivated limbal epithelial transplantation, and the corneal epithelial layer migrates on its surface. As in the ‘‘graft’’ transplantation method, the amniotic membrane is covered by the epithelial layer after epithelialization.18 As suggested by Tsai et al,19 the amniotic membrane has a slow absorption rate when this transplantation method is used. By electronic microscopy and histologic examination, Resch et al20 discovered that the amniotic membrane persisted in the corneal stroma for up to 79 months. Whether the amniotic membrane can exist in the corneal stroma for a longer period and the time of its final disappearance after cultivated limbal epithelial 1196
transplantation need further follow-up. In contrast, the amniotic membrane could not be observed at 8–10 months after cultivated limbal epithelial transplantation in the 4 eyes suffering immune rejection in our series. Confocal microscopy showed a large amount of inflammatory cell infiltration in the corneal stroma beneath the amniotic membrane, but this abnormality was not found in eyes with stable ocular surfaces. Therefore, we hypothesized that the chronic inflammation on the ocular surface caused by immune rejection was an important factor in accelerating the disappearance of the amniotic membrane.21–25 The amniotic membrane is translucent. Gris et al17 believed that the persistent presence of the amniotic membrane in the corneal stroma affected corneal transparency. In this study, at 7–10 days after cultivated limbal epithelial transplantation, the amniotic membrane was grayish-white and cloudy and adhered tightly to the corneal surface. Due to the combination of surgical stimulation and inflammation, the corneal stroma exhibited edema, cloudiness and decreased transparency. At 1 month, no amniotic membrane tissues but subepithelial ground-glass opacity was observed on the ocular surface. The stromal edema had subsided, showing improved transparency. The BCVA was significantly improved. At 2 months, fine gridlike cloudiness was detected beneath the corneal epithelium in patients with stable ocular surfaces, and it gradually faded during the follow-up period. The homogeneous, reflectively enhanced acellular structure, which was the unabsorbed amniotic membrane tissues, was visible only by confocal microscopy at 1 year. The BCVA was not
AMERICAN JOURNAL OF OPHTHALMOLOGY
DECEMBER 2014
significantly different from that at 1 month. This indicates that the presence of stable amniotic membrane exerts no effect on the corneal stromal transparency or visual acuity. In contrast, corneal cloudiness and neovascularization occurred again in the 4 eyes suffering immune rejection. The BCVA substantially decreased during the follow-up. This suggests that much attention is needed to prevent and treat immune rejection after cultivated limbal epithelial transplantation.14 In infective corneal diseases, the transplantation of a multilayer amniotic membrane can increase the thickness of the corneal stroma and avoid complications such as ulcer perforation.26–30 In this study, corneal OCT showed gradual thinning of the amniotic membrane in the 12
eyes with stable ocular surfaces. Discontinuity of the amniotic membrane was observed at 4 months after cultivated limbal epithelial transplantation and became more evident at 1 year. The corneal stromal thickness did not change significantly. This is most likely related to the use of the single-layer, thin amniotic membrane. Amniotic membrane is an ideal vehicle for the in vitro cultivation and transplantation of limbal epithelial stem cells. It can survive for at least 1 year in the cornea of a patient with a stable ocular surface, without impact on the visual acuity, corneal stromal transparency or thickness. Chronic inflammation and neovascularization of the ocular surface can accelerate the absorption of the amniotic membrane and lead to decreased corneal transparency.
ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST, and the following were reported. This study was supported by the National Basic Research Program of China (2013CB967004) and the Specialized Research Fund for the Doctoral Program of Higher Education (20113706110004). Involved in Design and conduct of study (X.Q.); Collection of data (J.W., D.S.); Management, analysis and interpretation of data (X.Q., J.W., Q.Z.); Preparation of manuscript (X.Q.); Review and approval of manuscript (L.X.). The authors thank Ping Lin, Shandong Eye Institute, for her linguistic assistance.
REFERENCES 1. Koizumi N, Inatomi T, Suzuki T, Sotozono C, Kinoshita S. Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology 2001;108(9): 1569–1574. 2. Koizumi N, Inatomi T, Suzuki T, Sotozono C, Kinoshita S. Cultivated corneal epithelial transplantation for ocular surface reconstruction in acute phase of Stevens-Johnson syndrome. Arch Ophthalmol 2001;119(2):298–300. 3. Nakamura T, Sotozono C, Bentley AJ, et al. Long-term phenotypic study after allogeneic cultivated corneal limbal epithelial transplantation for severe ocular surface diseases. Ophthalmology 2010;117(12):2247–2254. 4. Dua HS, Gomes JA, King AJ, Maharajan VS. The amniotic membrane in ophthalmology. Surv Ophthalmol 2004;49(1): 51–77. 5. Koizumi NJ, Inatomi TJ, Sotozono CJ, Fullwood NJ, Quantock AJ, Kinoshita S. Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res 2000; 20(3):173–177. 6. Meller D, Pires RT, Tseng SC. Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures. Br J Ophthalmol 2002;86(4): 463–471. 7. Burman S, Tejwani S, Vemuganti GK, Gopinathan U, Sangwan VS. Ophthalmic applications of preserved human amniotic membrane: a review of current indications. Cell Tissue Bank 2004;5(3):161–175. 8. Dua HS, Joseph A, Shanmuganathan VA, Jones RE. Stem cell differentiation and the effects of deficiency. Eye 2003; 17(8):877–885. 9. Shortt AJ, Secker GA, Notara MD, et al. Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results. Surv Ophthalmol 2007;52(5): 483–502.
VOL. 158, NO. 6
10. Lee S, Tseng SC. Amniotic membrane transplantation for persistent epithelial defects with ulceration. Am J Ophthalmol 1997;123(3):303–312. 11. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000;343(2):86–93. 12. Nishida K, Yamato M, Hayashida Y, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 2004; 351(12):1187–1196. 13. Zhou Q, Yang L, Wang Y, et al. TGFbeta mediated transition of corneal fibroblasts from a proinflammatory state to a profibrotic state through modulation of histone acetylation. J Cell Physiol 2010;224(1):135–143. 14. Qi X, Xie L, Cheng J, Zhai H, Zhou Q. Characteristics of immune rejection after allogeneic cultivated limbal epithelial transplantation. Ophthalmology 2013;120(5):931–936. 15. Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 2010;363(2):147–155. 16. Chen Y, Yan XM, Wu HR, Rong B. An experimental study on the fate of the amniotic membrane after amniotic membrane transplantation for acute alkaline burn of rat cornea. Zhonghua Yan Ke Za Zhi 2012;48(1):27–32. 17. Gris O, Wolley-Dod C, Gu¨ell JL, et al. Histologic findings after amniotic membrane graft in the human cornea. Ophthalmology 2002;109(3):508–512. 18. Maharajan VS, Shanmuganathan V, Currie A, Hopkinson A, Powell-Richards A, Dua HS. Amniotic membrane transplantation for ocular surface reconstruction: indications and outcomes. Clin Exp Ophthalmol 2007;35(2):140–147. 19. Tsai RJ, Tsai RY. Ex vivo expansion of corneal stem cells on amniotic membrane and their outcome. Eye Contact Lens 2010;36(5):305–309. 20. Resch MD, Schlo¨tzer-Schrehardt U, Hofmann-Rummelt C, et al. Integration patterns of cryopreserved amniotic
AMNIOTIC MEMBRANE IN LIMBAL EPITHELIAL TRANSPLANTATION
1197
21.
22. 23.
24.
25.
membranes into the human cornea. Ophthalmology 2006; 113(11):1927–1935. Gris O, Lo´pez-Navidad A, Caballero F, del Campo Z, Ada´n A. Amniotic membrane transplantation for ocular surface pathology: long-term results. Transplant Proc 2003;35(5): 2031–2035. Bourne WM. Cellular changes in transplanted human corneas. Cornea 2001;20(6):560–569. Hollingsworth JG, Efron N, Tullo AB. A longitudinal case series investigating cellular changes to the transplanted cornea using confocal microscopy. Cont Lens Anterior Eye 2006; 29(3):135–141. Chen M, Gong L, Xu J, Zhu W, Devine EE. Ultrastructural and in vivo confocal microscopic evaluation of interface after Descemet’s stripping endothelial keratoplasty in rabbits. Acta Ophthalmol 2012;90(1):e43–47. Abdelkader A, Kaufman HE. Descemetic versus preDescemetic lamellar keratoplasty: clinical and confocal study. Cornea 2011;30(11):1244–1252.
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AMERICAN JOURNAL OF OPHTHALMOLOGY
DECEMBER 2014
Biosketch Dr Xiaolin Qi is an attending ophthalmologist at the Shandong Eye Hospital of Shandong Eye Institute. She received her master’s degree and doctoral degree in ophthalmology from Qingdao University. Dr Qi is engaged in clinical work of corneal disease.
VOL. 158, NO. 6
AMNIOTIC MEMBRANE IN LIMBAL EPITHELIAL TRANSPLANTATION
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